Human cytochrome P450IIA3: cDNA sequence, role of the enzyme in the metabolic activation of promutagens, comparison to nitrosamine activation by human cytochrome P450IIE1

We report that, in a human cell line, human cytochrome P450IIA3 is capable of metabolizing aflatoxin B1, benzo[a]-pyrene, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to cytotoxic and mutagenic species. Cytochrome P450IIA3-mediated activation of NDMA and NDEA was compared with human cytochrome P450IIE1-mediated activation in the same cell system. P450IIE1 was more effective at activating NDMA than P450IIA3, while P450IIA3 was more effective at activating NDEA than P450IIE1. Whole cells and microsomal fractions obtained from control cells and from cells expressing the P450IIA3 cDNA were characterized for expression of P450IIA3. Microsomal coumarin 7-hydroxylase activity was some 40 times greater in the transfected cells than in the control cells and was catalyzed by a protein that was immunochemically related to the rat liver cytochrome P450IIA gene family. Immunoblot analysis demonstrated that this protein was readily detectable in transfected cells but barely detectable in control cells. We also report the DNA and deduced amino acid sequence of the P450IIA3 cDNA isolate used in this study. Our isolate encodes a protein 489 amino acids that is five amino acids shorter at the N terminus but otherwise identical to a previously reported human P450IIA3 cDNA sequence.     

Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair-proficient CHO cells.

Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide-excision-repair-deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair-competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair-competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair-deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 microM, while the repair-proficient 5P1R3 cells showed no toxicity up to 60 microM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.     

Stable expression of human cytochrome P450IA2 cDNA in a human lymphoblastoid cell line: role of the enzyme in the metabolic activation of aflatoxin B1

A human lymphoblastoid cell line stably expressing a human cytochrome P450IA2 cDNA was developed. This recombinant cell line displayed P450IA2 protein and estradiol 2-hydroxylase activity, neither of which was detected in the parental cell line. The recombinant cell line was also approximately 1000-fold more sensitive to the cytotoxicity and mutagenicity of the carcinogenic mycotoxin aflatoxin B1 than was the parent cell line. The increase in mutagenicity was supported by a corresponding increase in the level of aflatoxin B1 binding to DNA in cells expressing P450IA2 relative to control cells.     

Heterologous expression of rat P450 2E1 in a mammalian cell line: in situ metabolism and cytotoxicity of N-nitrosodimethylamine

GM0637, a human fibroblast cell line, was transfected with pCMV2E1, an expression vector containing the full length cDNA for rat cytochrome P450 2E1 (P450 2E1), and with pCMVneo, which contained vector alone, and the selected clones were designated GM2E1 and GMneo, respectively. Western blot analysis showed that GM2E1, but not GMneo, expressed a protein that reacted with anti-human P450 2E1 antibody. The 7- ethoxycoumarin O-deethylase,p-nitrophenol hydroxylase, and N- nitrosodimethylamine (NDMA) demethylase activities of the P450 in these cells were measured in monolayer cell cultures without preparing microsomes. Exposure of the GM2E1 cells to NDMA for 4 days caused severe decreases in cell viability, as determined by crystal violet uptake, and showed a sigmoidal dose-response curve with a median lethal dose of 17 microM. In contrast, the viability of GMneo cells was not altered by NDMA even at concentrations up to 10 mM. Time- and concentration-dependent methylation of DNA, RNA and protein by [14C]NDMA was only observed in cells expressing P450 2E1. Inhibitors of P450 2E1 activity such as ethanol, 4-methylpyrazole, and isoniazid caused a 90% decrease in the methylation of cellular macromolecules and also completely protected the cells against NDMA-mediated toxicity. The cytotoxicity due to exposure to NDMA was partially inhibited by antioxidants such as N-acetylcysteine, ascorbic acid, butylated hydroxyanisole and N-t-butyl-alpha-phenylnitrone but was not potentiated upon glutathione depletion. These results document the ability of rat P450 2E1 to metabolize NDMA to toxic reactive intermediates and demonstrate that this cell line provides a useful model for studying the mechanisms of metabolism-mediated toxicity and carcinogenesis.      

Elevated cell migration, invasion and tumorigenicity in human KB carcinoma cells transfected with COX-2 cDNA

In order to investigate the involvement of cyclooxygenase (COX)-2 in cell growth and invasion of oral cancer, a human epidermoid carcinoma cell line KB minimally expressing COX-2 protein was transfected with COX-2 cDNA and these activities were compared with mock-transfected KB in vitro and in vivo. KB/COX-2 clones showed a similar growth rate in vitro compared to KB/neo clones, but demonstrated significantly increased PGE2 production, cell migration and invasion. These KB/COX-2 clones markedly expressed MMP-9, pro-MMP-2 and activated-MMP-2 as compared to KB/neo clones in gelatin zymography. Western blot analysis showed that expression of MT1-MMP, Rho and Rac 1 in KB/COX-2 clones were stronger than that in KB/neo clones, but expression of TIMP-1 and TIMP-2 were weaker in KB/COX-2 clones than in KB/neo clones. When these cells were inoculated subcutaneously into nude mice, tumorigenicity and tumor growth were significantly elevated in KB/COX-2 tumors than in KB/neo tumors, and the gelatinase activity was much stronger in KB/COX-2 tumor tissues than in KB/neo tumor tissues in film in situ zymography. The orthotopic inoculation of cells to the oral floor showed that local invasion was pronounced in KB/COX-2 tumors. These results indicated that overexpression of COX-2 elevated tumorigenicity, tumor growth and invasion of human KB carcinoma cells via up-regulated MMP and Rho family small GTPases and down-regulated TIMP activities.     

大肠杆菌嘌呤核苷磷酸化酶基因真核表达载体的构建及其在MKN-45细胞中的表达

目的克隆大肠杆菌嘌呤核苷磷酸化酶(EPNP)基因并构建真核表达载体pcDNA3.1-EPNP,将重组质粒转染MKN-45细胞获得高表达大肠杆菌PNP基因的MKN-45细胞克隆。方法根据Gen-bank中大肠杆菌PNP基因的核苷酸序列,设计并合成了一对引物,以大肠杆菌基因组DNA为模板,进行PCR扩增,并将扩增产物定向插入pMD-18T克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pcDNA3.1,利用脂质体将重组质粒转染MKN-45细胞。结果PCR扩增出716bp大小的片段,测序结果与已报道的EPNP基因序列一致;亚克隆经酶切鉴定正确;RT-PCR证明经G418筛选得到的转基因MKN-45细胞克隆中存在大量EPNPmRNA,前体药MePdR对转染EPNP的MKN-45细胞有较强的细胞毒作用。结论成功构建了大肠杆菌PNP基因的真核表达载体,获得了稳定表达大肠杆菌PNP基因的MKN-45细胞克隆,为PNP/MepdR自杀基因系统在胃癌基因治疗中的应用奠定了良好的基础。     

Transfection of a rat cytochrome P450b cDNA into C3H10T1/2CL8 mouse embryo fibroblasts

A cDNA clone of a rat cytochrome P450b gene was used to construct an expression vector driven by an SV40 promoter and containing a G418-resistance selectable marker. This bifunctional plasmid (pJRSL100) was transfected into the C3H 10T1/2CL8 mouse embryo fibroblast cell line. G418-resistant clones were selected and tested for enhanced sensitivity to the carcinogen 2-acetylaminofluorene (2-AAF), a compound that does not normally induce cytotoxicity or morphological transformation in these cells. One subclone, 19P450b-4, exhibited an increased cytotoxic response to 2-AAF compared to the parental C3H10T1/2CL8 cells. DNA analyses of this subclone showed increased number of copies of the cytochrome P450b and the appearance of unique restriction fragment bands relative to parental and control transfected cells. This subclone also exhibited increased levels of mRNA complementary to the P450b cDNA. Metabolism studies of 2-AAF in this subclone demonstrated an increase in the C-hydroxylated metabolites 1-, 3-, 5/9-, and 7-hydroxy-AAF compared with parental C3H 10T1/2CL8 cells. The results indicate that C3H 10T1/2CL8 cells can be transfected with gene/cDNAs to increase their metabolic competency and that such transfection may enhance the usefulness of the C3H 10T1/2CL8 cells in studies on chemically induced cytotoxicity and morphological transformation.     

stathmin基因的RNA干涉抑制人白血病K562细胞体外增殖的作用

目的：研究RNA干涉（RNA interference，RNAi）抑制stathmin基因表达后对人白血病K562细胞体外增殖的作用。方法：将合成的寡核苷酸链退火形成双链，连接入经BamHI和HindIII双酶切后的pSilencer4．1-CMV neo真核表达载体。酶切及测序鉴定。脂质体法转染重组质粒入K562细胞，RT—PCR检测其对mRNA的干涉效果；MTT比色法检测K562细胞体外增殖能力。结果：经酶切及测序鉴定，成功构建重组质粒。RT—PCR显示所构建的干涉载体成功地抑制了目的基因的转录，同时细胞增殖活性降低。结论：RNA干涉成功抑制stathmin基因表达并使人白血病K562细胞体外增殖活性明显降低。     

survivin启动子调控siRNA真核表达载体的构建及对HeLa细胞中stathmin基因的干涉作用

目的:探讨survivin基因启动子调控的siRNA真核表达载体对HeLa细胞中stathmin基因表达的肿瘤特异性封闭作用。方法:合成针对人stathmin基因的siRNA cDNA寡核苷酸链退火形成双链,连接入经BamHI和HindIII双酶切后的pSilencer4.1-CMV neo真核表达载体。PCR扩增suvivin基因启动子并测序,用EcoRI和BamHI分别双酶切,连接至经相同内切酶消化的载体,获得survivin启动子调控的siRNA真核表达载体,酶切及测序鉴定。脂质体法转染重组质粒入HeLa细胞,G418筛选。RT-PCR扩增stathmin基因,检测其对stathmin基因表达的干涉效果;流式细胞仪分析转染后HeLa细胞增殖周期的改变。结果:经酶切及测序鉴定,成功构建重组质粒;RT-PCR结果显示所构建的干涉载体可有效封闭stathmin基因表达;流式细胞仪分析结果显示,Hale细胞在stathmin siRNA作用下G2/M期细胞的比例明显增加。结论:survivin基因启动子调控的siRNA真核表达载体可有效地封闭HeLa细胞中stathmin基因表达并使HeLa细胞阻断于G2/M期,为以stathmin基因为靶点的恶性肿瘤生物治疗奠定了理论基础。     

人ＳＴＡＴ３基因ｓｉＲＮＡ真核表达质粒的构建及鉴定

目的：构建针对人ＳＴＡＴ３基因的ｓｉＲＮＡ真核表达质粒,检测其在细胞水平对ＳＴＡＴ３基因表达的抑制效果。方法：用ＤＮＡ重组技术将针对人ＳＴＡＴ３基因ｍＲＮＡ序列不同位点设计的３个ｓｉＲＮＡ序列克隆到真核表达质粒ｐＲＮＡＴ-Ｕ６.１／ｎｅｏ中构建重组体ｐＲＮＡＴ-Ｕ６.１-ｓｉＲＮＡ,重组质粒经ＰＣＲ检测及测序分析,用脂质体转染重组质粒至人食管癌Ｅｃａ-１０９细胞,Ｇ４１８筛选获得阳性克隆,ＲＴ-ＰＣＲ和Ｗｅｓｔｅｒｎｂｌｏｔ检测ＳＴＡＴ３基因ｍＲＮＡ和蛋白的表达,筛选最佳沉默效率的ｓｉＲＮＡ。结果：ＰＣＲ检测及测序分析结果均提示重组质粒构建正确。ＲＴ-ＰＣＲ和Ｗｅｓｔｅｒｎｂｌｏｔ检测证实ｐＲＮＡＴ-Ｕ６.１-ｓｉＲＮＡ３具有最佳的沉默效率。结论：成功构建人ＳＴＡＴ３基因ｓｉＲＮＡ真核表达质粒,并证实其能够从ｍＲＮＡ和蛋白水平抑制ＳＴＡＴ３基因的表达。     

Selective mutagenic activation by cytochrome P3-450 of carcinogenic arylamines found in foods

Heterocyclic arylamines found in cooked foods including fish and beef are potent mutagens and carcinogens. The purpose of this investigation was to determine the specificity of cytochromes P1-450 and P3-450 toward the metabolic activation of these arylamines. We used a novel mutagenicity test system which combined human cells expressing either recombinant cytochrome P1-450 or P3-450 with Salmonella typhimurium to score mutants. Cytochrome P3-450, a single isoform of the cytochrome P-450 supergene family, bioactivated these food mutagens. Cytochrome P1-450 showed little or no activation of these arylamines but was the isoform predominantly responsible for the activation of the aromatic hydrocarbon benzo[a]pyrene-7,8-diol. This assay system should serve to define the specificities of individual cytochromes P-450 in the metabolic activation of carcinogens.     

Substrate specificity and alkyl group selectivity in the metabolism of N-nitrosodialkylamines

Metabolic activation may be a key step in determining the tissue specificity of carcinogenic nitrosamines. In previous work, we characterized P450IIE1 (an acetone/ethanol-inducible form of cytochrome P-450) as the major enzyme for the metabolic activation of N-nitrosodimethylamine. In this work, we investigated the metabolism of other N-nitrosodialkylamines in rat liver microsomes and in reconstituted monooxygenase systems containing purified cytochrome P-450 isozymes. The enzyme specificities in the metabolism of N-nitrosoethylmethylamine and N-nitrosodiethylamine were similar to those of N-nitrosodimethylamine; i.e., these substrates were more efficiently metabolized by acetone- or ethanol-induced microsomes than by other types of microsomes. However, substituting one methyl group with a benzyl or butyl group, as in N-nitrosobenzylmethylamine or N-nitrosobutylmethylamine (NBMA), substantially changed the enzyme specificity. P450IIE1 efficiently catalyzed the demethylation but not the debutylation of NBMA, whereas P450IIB1 (a phenobarbital-inducible form) efficiently catalyzed both the debutylation and demethylation reactions. In the demethylation of NBMA by P450IIE1, the addition of cytochrome b5 markedly increased the activity at low but not at high substrate concentrations, suggesting a decrease in Km value. This effect, however, was not observed in the debutylation of NBMA by P450IIE1 or P450IIB1, and in the demethylation of NBMA by P450IIB1. These studies demonstrate the substrate specificity and alkyl group selectivity in the metabolism of nitrosamines by cytochrome P-450 isozymes.     

Expression of human cytochrome P450 1A1 in DNA repair deficient and proficient human fibroblasts stably transformed with an inducible expression vector

Cytochromes P450 catalyze the bioactivation of many carcinogens.In particular, cytochrome P450 1A1 (CYP1A1) catalyzes the conversionof polycyclic aromatic hydrocarbons, such as benzo[a]pyrene,into potent mutagenic agents. Human skin fibroblasts, both DNArepair deficient (xeroderma pigmentosum group A: XPA) and DNArepair normal have been co-transformed with a chimeric geneconstruct containing human CYP1A1 coding sequences controlledby the cadmium (Cd) ion inducible mouse metallothionein-I promoterand pRSV-NEO, a dominant selectable marker for G418 resistance.Individual G418 resistant colonies were cloned and analyzedfor Cd inducible CYP1A1 activity. Six clones of DNA repair deficientcells and five clones of DNA repair proficient cells have beenisolated which express Cd inducible CYP1A1. Benzo[a]pyrene-Aww-7,8-diol (BPD) is cytotoxic in Cd induced CYP1A1 expressing cells.The cytotoxkity can be inhibited by 10 µM     

Development of a human cell line by selection and drug-metabolizing gene transfection with increased capacity to activate promutagens

We have isolated a human lymphoblastoid cell line with higher levels of native cytochrome P450IA1 activity and by DNA transfection introduced human cDNAs for a putative cytochrome P450IIA2 and epoxide hydrolase (E.C. 3.3.2.3). The resultant cell line, designated MCL-1, was substantially more sensitive to the mutagenicity of dimethylnitrosamine and benzo[a]pyrene than the AHH-1 cell line and was found to have increased metabolism of benzo[a]pyrene to dihydrodiols. The increase in native cytochrome P450IA1 activity was achieved by mutation and selection based on resistance to the phototoxicity of benzo[ghi]perylene. One resistant clone, designated L3, was used for subsequent studies. Two complete cDNAs, one encoding a putative cytochrome P450IIA2 and the other a microsomal epoxide hydrolase, were isolated from a human liver cDNA library. After introduction of the cDNAs into an expression vector and transfection into AHH-1 cells, gene expression was detected at the level of enzyme activity (epoxide hydrolase) or by increased sensitivity to dimethylnitrosamine cytotoxicity/mutagenicity (putative P450IIA2). A vector containing both cDNAs was then constructed and transfected into L3 cells to produce MCL-1 cells. The potential usefulness of drug-metabolizing gene transfection and of the MCL-1 cell line, in particular, for genetic toxicity testing is discussed.     

Association between restriction fragment length polymorphism of the human cytochrome P450IIE1 gene and susceptibility to lung cancer.

Cytochrome P450IIE1 (P450IIE1) is involved in metabolic activation of carcinogenic nitrosamines, aniline and benzene. We detected a restriction fragment length polymorphism of the human P450IIE1 gene with the restriction endonuclease DraI. The population was thus divided into three genotypes, namely, heterozygotes (CD) and two forms of homozygotes (CC and DD). The distribution of these genotypes among lung cancer patients differed from that among controls with statistical significance of P less than 0.05 (chi 2 = 7.01 with 2 degrees of freedom). This result strongly suggests that host susceptibility to lung cancer is associated with the DraI polymorphism of the P450IIE1 gene.     

Molecular basis for the sex-related difference in renal N-nitrosodimethylamine demethylase in C3H/HeJ mice

Previous work with rat and rabbit liver enzymes has demonstrated that cytochrome P450IIE1 is responsible for the metabolism of N-nitrosodimethylamine (NDMA), a widely occurring carcinogen. The present study demonstrated that a similar enzyme also exists in the mouse kidney and is regulated by testosterone. These results can account for the reported sex-related difference in the renal metabolism of NDMA in mouse strains such as C3H/HeJ. NDMA demethylase activities (expressed as pmol/min/mg protein) in kidney microsomes of female and male C3H/HeJ mice were 3.0 +/- 0.7 and 51.9 +/- 11.2, respectively. After testosterone treatment (500 mg/kg b.w. in olive oil, s.c.) for 2 days, the renal NDMA demethylase activity of the female mice was elevated 17-fold. The difference and change in NDMA demethylase activity were accompanied by corresponding differences and changes in P450IIE1 as quantified by immunoblot analysis (using antibodies prepared against rat P450IIE1) as well as in the mRNA level for P450IIE1 as determined by Northern and slot blot analyses (using a cDNA probe containing the coding sequence of rat P450IIE1 gene). Based on gel electrophoresis, the molecular weight of mouse renal P450IIE1 was 52,000 and the size of mouse renal P450IIE1 mRNA was approximately 1.8 kilobases; both were similar to those found in rat liver and kidney. Renal P450IIE1 mRNA levels in female, male, and testosterone-treated female mice were at a ratio of 1:22:20. On the other hand, this testosterone-related difference was not observed in hepatic P450IIE1. In liver microsomes, there were no significant differences in NDMA demethylase activity, P450IIE1 content, and P450IIE1 mRNA level between male and female mice or between untreated and testosterone-treated female mice. The apparent Km value of NDMA demethylase in mouse kidney microsomes (22 to 27 microM NDMA) were similar to that in rat liver microsomes. Renal NDMA demethylase activity was inhibited by a monoclonal antibody prepared against rat P450IIE1. These results suggest that mouse renal P450IIE1 is similar to rat P450IIE1 and is responsible for the low Km form of NDMA demethylase activity. Nevertheless, only the mouse renal enzyme is regulated by testosterone.     

Association between Restriction Fragment Length Polymorphism of the Human Cytochrome P450IIE1 Gene and Susceptibility to Lung Cancer

Cytochrome P450IIE1 (P450IIE1) is involved in metabolic activation of carcinogenic nitrosamines, aniline and benzene. We detected a restriction fragment length polymorphism of the human P450IIE1 gene with the restriction endonuclease Oral. The population was thus divided into three genotypes, namely, heterozygotes (CD) and two forms of homozygotes (CC and DD). The distribution of these genotypes among lung cancer patients differed front that among controls with statistical significance of P< 0.05 (x²=7.01 with 2 degrees of freedom). This result strongly suggests that host susceptibility to lung cancer is associated with the Dral polymorphism of the P450IIE1 gene.     

EJ/ras neoplastic transformation of simian virus 40-immortalized human uroepithelial cells: a rare event

To determine if expression of mutant p21 ras could convert Simian Virus 40-immortalized human uroepithelial cell line (SV-HUC) to tumorigenicity, SV-HUC cells were transfected with pSV2-neo (a neomycin-resistant gene) or PREJ/ras (c-HA-ras-1 with the 12th codon mutation and neo). Seven independent G418-resistant clones (A----G) were isolated from each group (SV-HUC/ras and SV-HUC/neo). SV-HUC/ras clones were morphologically altered, while SV-HUC/neo clones retained a typical SV-HUC epithelial morphology. Electrophoretic analysis of immunoprecipitated ras proteins detected altered p21 ras protein in four of seven SV-HUC/ras clones at passage (P)2 and in five of seven clones at P12 posttransfection. The relative levels of ras p21 differed among the clones and appeared to increase with passage in culture. RNA and DNA dot blot analyses showed that clones with more abundant mutant p21 also had higher ras RNA levels and, in one case, increased ras gene copy number. No altered ras protein was detected in any SV-HUC/neo clones. ras- and neo-transfected clones were tested for tumorigenicity at P2 posttransfection and again at P12 by four s.c. inoculations each into athymic nude mice. None of 56 inoculations of SV-HUC/neo clones was tumorigenic. None of the SV-HUC/ras clones at P2 gave rise to tumors at all four injection sites. However, two ras-transfected clones, SV-HUC/ras-B and SV-HUC/ras-F, produced one tumor each. One clone, SV-HUC/ras-D which produced abundant mutant p21, was negative when inoculated at P2, but produced tumors in four of four sites when reinoculated after ten passages in vitro. All tumorigenic clones had detectable levels of mutant ras p21. However, the relative levels of altered p21 ras protein among the SV-HUC/ras clones did not directly predict their tumorigenic potential, as several nontumorigenic SV-HUC/ras clones had protein levels equal to or higher than the most tumorigenic clone (SV-HUC/ras-D at P12). Cell lines established from the tumor explants exhibited higher ras gene copy numbers, higher RNA levels, and more abundant p21 than was seen in the clones at the time of inoculation. Therefore, increases in ras protein abundance occurred during tumor formation in vivo, as well as during passage of cells in culture, and such cells apparently had a selective growth advantage. However, expression of abundant mutant ras protein was not in itself sufficient for neoplastic transformation of SV-HUC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reductive metabolism of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) and the induction of unscheduled DNA synthesis in rat and human derived cell lines

The cellular and microsomal reductive metabolism of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) was studied aerobically and anaerobically in rat (JB1, BL8, Walker) mouse (3T3) and human (HepG2) derived cell lines. In all cases rates or reduction to the mono-N-oxide (SR 4317) were greater under anaerobic than aerobic conditions when determined using HPLC with UV and fluorescence detection. Of the rat derived cells, a transformed liver epithelial line (JB1) showed the greatest cellular and microsomal rates of reduction while Walker 256 carcinoma cells showed the lowest. Microsomal reduction of SR 4233 was the highest in preparations from rat liver but carbon monoxide did not inhibit the reaction, suggesting cytochrome P450 was not involved. Purified cytochrome P450 reductase could carry out the reduction of SR4233. Rates of reduction of SR4233 in microsomal preparations from the cell lines and in rat liver nuclei correlated with activities of cytochrome P450 reductase. Activation of SR 4233 to cause cellular DNA damage was measured using unscheduled DNA synthesis as an index. Under anaerobic conditions, SR 4233 caused a dose-dependent increase in unscheduled DNA synthesis. Only a slight increase occurred following aerobic incubation. No increase was seen using the reduced metabolites SR 4317 or SR 4330. Of the cell lines studied, Walker cells showed the highest induction of unscheduled DNA synthesis. It was concluded that rates of reduction of SR 4233 did not necessarily reflect the potential DNA-damaging effects of this compound.     

Different malignant phenotypes induced in a stable, subdiploid, benign epithelial clone by DNA transfection.

Progression to malignancy in carcinomas has been studied in a stable, benign, subdiploid, cloned epithelial cell line (A5P/B10) sensitive to Geneticin at 100 micrograms/ml. A total of 28 cell lines were selected for Geneticin - resistance and inoculated into the footpads of syngeneic animals following co-transfection with pSV2neo and genomic DNA, or transfection with plasmid constructs containing neo and the activated Ha-ras oncogene. The behavior of 12 cell lines cotransfected with normal genomic DNA and inoculated into 146 footpads was the same as the A5P/B10 cells. Low grade primary tumors were produced in 122 footpads by 13 cell lines transfected with Ha-ras, and a proportion (61/122) produced well-differentiated lymph node metastases. One of 3 cell lines cotransfected with genomic DNA from a malignant cell line (BC1) produced 8 anaplastic primary tumors with anaplastic metastases. Cell lines from lymph nodes involved by these anaplastic tumors were sensitive to Geneticin, and genomic DNA from 2 clones of these cells failed to produce a malignant phenotype when co-transfected into the A5P/B10 cells. These results indicated that the progression to a malignant phenotype induced in benign cells from a spontaneous epithelial tumor by co-transfection with genomic DNA from malignant cells was different from that induced by the ras oncogene.